¹Cancer Biology Program, Department of Oncology, The Sidney Kimmel Comprehensive Cancer, Center at Johns Hopkins, Baltimore, MD, USA

Correspondence: Dr JG Herman, Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Bunting Blaustein Cancer Research Building, 1650 Orleans Street, Suite 543, Baltimore, MD 21231-1000, USA. E-mail: hermanji@jhmi.edu

Received 16 August 2006; Revised 13 December 2006; Accepted 25 January 2007; Published online 26 March 2007.

Abstract

Methylation-specific polymerase chain reaction (PCR) (MSP) is frequently used to study gene silencing by promoter hypermethylation. However, non-specific primer design can lead to false-positive detection of methylation. We present a novel, web-based algorithm for the design of primers for bisulfite-PCRs (MSP, sequencing, COBRA and multiplex-MSP), allowing the determination of a specificity score, which is based on the thermodynamic characteristics of the primer 3'-end. PCR amplification with primers not reaching a high specificity score can result in false-positive findings. We used MSPprimer to design MSP primers for analysis of the ATM promoter. In 37 non-small cell lung cancer (NSCLC) samples and 43 breast cancer samples no promoter methylation was detected. Conversely, published MSP primers not reaching the required specificity score led to non-specific amplification of DNA not converted by bisulfite. The result was a false-positive incidence of ATM promoter methylation of 24% in NSCLC and 48% in breast cancers, similar to published studies. This highlights the critical need for specific primer design for MSP. MSPprimer is a convenient tool to achieve this goal, which is available free of charge to the scientific community.